Wanlapa Roobsoong

Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

Determination of the Plasmodium vivax schizont stage proteome

With the genome of the malaria parasite Plasmodium vivax sequenced, it is important to determine the proteomes of the parasite in order to assist efforts in antigen and drug target discovery. Since a method for continuous culture of P. vivax parasite is not available, we tried to study the proteome of the erythrocytic stages using fresh parasite isolates from patients. In schizont-enriched samples, 316 proteins were confidently identified by tandem mass spectrometry. Almost 50% of the identified proteins were hypothetical, while other major categories include proteins with binding function, protein fate, protein synthesis, metabolism and cellular transport. To identify proteins that are recognized by host humoral immunity, parasite proteins were separated by two-dimensional gel electrophoresis and screened by Western blot using an immune serum from a P. vivax patient. Mass spectrometry analysis of protein spots recognized by the serum identified four potential antigens including PV24. The recombinant protein PV24 was recognized by antibodies from vivax malaria patients even during the convalescent period, indicating that PV24 could elicit long-lasting antibody responses in P. vivax patients.

Infectivity of symptomatic and asymptomatic Plasmodium vivax infections to a Southeast Asian vector, Anopheles dirus

Plasmodium vivax is now the predominant species causing malarial infection and disease in most non-African areas, but little is known about its transmission efficiency from human to mosquitoes. Because the majority of Plasmodium infections in endemic areas are low density and asymptomatic, it is important to evaluate how well these infections transmit. Using membrane feeding apparatus, Anopheles dirus were fed with blood samples from 94 individuals who had natural P. vivax infections with parasitemias spanning four orders of magnitude. We found that the mosquito infection rate was positively correlated with blood parasitemia and that infection began to rise when parasitemia was >10parasites/μl. Below this threshold, mosquito infection is rare and associated with very few oocysts. These findings provide useful information for assessing the human reservoir of transmission and for establishing diagnostic sensitivity required to identify individuals who are most infective to mosquitoes.

Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement.

Significance Plasmodium vivax is responsible for the most widely distributed recurring human malaria infections whereas Plasmodium falciparum inflicts the most mortality and morbidity in human populations. Malaria parasites enter our blood cells by making proteins that recognize and bind to their cognate receptors on the red blood cell surface. Our research describes, to our knowledge, the first crystal structure of PvRBP2a, an erythrocyte-binding protein from P. vivax, which revealed a structural scaffold similar to that of PfRh5, the essential erythrocyte-binding protein in P. falciparum. Structural comparisons between PvRBP2a and PfRh5 provide an important foundation toward understanding how P. vivax and P. falciparum parasites use a homologous erythrocyte-binding protein family to engage alternate erythrocyte receptors and ultimately govern host cell specificity. Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short β-hairpin, and, although the structural fold is similar to that of PfRh5—the essential invasion ligand in Plasmodium falciparum—its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection.

Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins

ABSTRACT Members of the Plasmodium vivax reticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes by P. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure their in vivo transcript abundances in clinical P. vivax isolates. Like genes encoding related invasion ligands of P. falciparum, Pvrbp expression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion.

A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells.

BackgroundPlasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers.MethodsClinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One μL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intra-erythrocytic developmental stages.ResultsThe percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman’s rank correlation coefficient (R2 = 0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes.ConclusionsThe FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.

Fibrinogen domain of FREP1 is a broad spectrum malaria transmission-blocking vaccine antigen

FREP1 in mosquito midguts facilitates Plasmodium falciparum parasite transmission. The fibrinogen-like (FBG) domain of FREP1 is highly conserved (>90% identical) among Anopheles species from different continents, suggesting that anti-FBG antibodies may block malaria transmission to all anopheline mosquitoes. Using standard membrane-feeding assays, anti-FREP1 polyclonal antibodies significantly blocked transmission of Plasmodium berghei and Plasmodium vivax to Anopheles gambiae and Anopheles dirus, respectively. Furthermore, in vivo studies of mice immunized with FBG achieved >75% blocking efficacy of P. berghei to A. gambiae without triggering immunopathology. Anti-FBG serum also reduced >81% of P. falciparum infection to A. gambiae. Finally, we showed that FBG interacts with Plasmodium gametocytes and ookinetes, revealing the molecular mechanism of its antibody transmission-blocking activity. Collectively, our data support that FREP1-mediated Plasmodium transmission to mosquitoes is a conserved pathway and that targeting the FBG domain of FREP1 will limit the transmission of multiple Plasmodium species to multiple Anopheles species.

The In Vitro Invasion Inhibition Assay (IIA) for Plasmodium vivax

Plasmodium vivax is considered as the most widely distributed human malaria parasite outside Africa. Studies of P. vivax malaria have always been limited due to the lack of continuously in vitro-propagated parasite lines. Due to this limitation, studies on P. vivax have lagged behind that of P. falciparum, which is routinely maintained in in vitro blood-stage culture. This method allows for the short-term ex vivo culture of P. vivax blood stages and as such offers a wealth of opportunities to study the biology of the blood stages of the parasite. In this chapter we describe the in vitro erythrocyte invasion inhibition assay (IIA) for P. vivax, which can be used as a powerful tool for blood-stage vaccine screening. The major challenges of this assay are the purification of schizont-stage parasites and host reticulocytes. The purification methods for both P. vivax schizont-stage parasites and reticulocytes as detailed here have been developed and simplified. The protocols in this chapter have been optimized to ensure that IIA becomes a more feasible and reliable assay.

A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages

BackgroundPlasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available.ResultsHere, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite.ConclusionsThe α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.